Complex enzymes perform multi-step catalysis in biosynthetic pathways in which labile or insoluble intermediates are channeled or transported between active sites. This project continues the structural study of polyketide synthases and glutamine amidotransferases. Microbes produce an astounding variety of polyketide natural products, which are an important source of new bio-active molecules that can be exploited as lead compounds for development of new pharmaceuticals. In these mega-enzyme assembly lines, a carrier domain transports insoluble intermediates between enzyme active sites. I. The modular organization of polyketide synthases is well suited to development of hybrid chemical- enzymatic synthetic systems, but a fundamental understanding of module architecture is lacking. A polyketide synthase module is composed of a two-enzyme extension region and a one-, two- or three- enzyme modification region. Modifying regions are understood at the level of structures of individual catalytic domains, but not as working units. Crystal structures will be determined for modifying regions of four polyketide synthases and, secondarily, for full modules. These studies will provide a critical understanding of how the polyketide synthases function as efficient assembly lines. II. A second aim is focused on the polyketide synthase for curacin A, an anti-cancer lead, which performs the rare and interesting syntheses of a cyclopropane and a terminal alkene. The mechanism, product specificity and substrate range of key enzymes for these transformations will be probed through high-resolution crystal structures of substrate or analog complexes, following on identification of the biosynthetic steps during the current period. The result of these studies will be an understanding of how the curacin synthase carries out its unusual chemical transformations, and to what extent the catalytic domains can tolerate substrate variants. The ~20 glutamine amidotransferases are responsible for most of the nitrogen incorporation reactions in the biosynthesis of primary metabolites. They are bi-functional enzymes in which a glutaminase active site hydrolyzes glutamine to produce NH3, which is channeled through the enzyme to a synthase/synthetase active site for nucleophilic attack on an acceptor substrate. III. We will continue a long-standing research program on glutamine amidotransferases, focusing on substrate binding and catalytic coupling in pyridoxal phosphate synthase and in CTP synthetase. The goal is to visualize the dynamic structures in a variety of states pertinent to their function. The results will be applicable to studies of other amidotransferases and also will inform efforts to develop anti- microbial or anti-proliferative leads for some amidotransferases. PUBLIC HEALTH RELEVANCE: Living systems synthesize a number of complicated and interesting chemical compounds. The biosynthetic machinery for making these molecules is a potential target for engineering to make new molecules or for inhibition to block growth of pathogens or tumor cells. These studies will uncover details about how complicated biosynthetic machines function and will aid engineering and drug discovery efforts.